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The C. reinhardtii strain C9 (CC-408, wild … The C. reinhardtii strain C9 (CC-408, wild type mt-) and lrl1-1 were obtained from the Kyoto University C. reinhardtii mutant library. Screening of lrl1-1 from the Chlamydomonas mutant library was performed according to Gonzalez-Ballestar et al. 2014 . Briefly, several primers spanning the upstream region to the 3' end region of Cre03.g197100 were designed. The superpool library was used as the PCR template, and any amplified product from a primer pair that included a specific region of Cre03.g197100 and AphVIII were sequenced, and its positional tag was identified. Further DNA sequencing indicated that AphVIII-tag insertion was in the upstream region of Cre03.g197100, 115 bp upstream of the 5' untranslated region (UTR), which is predicted to be the promoter region. Liquid cultures were grown in an Erlenmeyer flask mixotrophically in Tris-acetate-phosphate (TAP) medium. For all cultures, cells were cultivated under continuous illumination at 20-40 umol m^-2 s^-1 and 100 rpm shaking at 25C. To induce P-starvation, mid-log phase cells (3-5 x10^6 cells/ml) were centrifuged at 2000 rpm for 5 min and washed twice in a P-free (TAP-P) medium. Potassium phosphate was replaced by 1.5 mM KCl in TAP-P medium. Cells were initially adjusted to 1x10^5 cells/ml on culture day 0. Cultured cells were collected by centrifugation, immediately frozen in liquid nitrogen, and stored at -80C until use. id nitrogen, and stored at -80C until use.
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